REVIEWS BIOLOGY, STRUCTURE AND MECHANISM OF P-TYPE ATPases Werner Kühlbrandt P-type ATPases are ion pumps that carry out many fundamental processes in biology and medicine, ranging from the generation of membrane potential to muscle contraction and the removal of toxic ions from cells. Making use of the energy stored in ATP, they transport specific ions across the cell membrane against a concentration gradient. Recent X-ray structures and homology models of P-type pumps now provide a basis for understanding the molecular mechanism of ATP-driven ion transport. MEMBRANE POTENTIAL In 1957, Jens Skou examined the effect of various universally accepted and remains useful for identify- The charge difference cations on a homogenate of leg nerves from crabs that ing particular states. A summary of the catalytic cycle (measured in mV) between the were caught on the shores of Denmark. The results — or Post–Albers cycle — is shown in BOX 1.In their two surfaces of a biological led him to suggest that MEMBRANE POTENTIAL is generated normal mode of action, P-type pumps use ATP to membrane that arises from the by a K+-stimulated ATPase — now known as the maintain an ion gradient across the cell membrane, different concentrations of ions + + + such as H+,Na+ or K+ on either Na /K -ATPase — that uses ATP to transport Na and but each step is reversible, so P-type ATPases can, in side. The Na+/K+-ATPase creates K+ ions across the axonal membrane1. Little did he principle, use a membrane potential to produce ATP. a membrane potential by using know that he had just discovered the first P-type For further information about the early literature on the energy stored in ATP to ATPase, and that this would win him the Nobel prize in P-type ATPases, see REF. 6. maintain a low concentration of Na+ and a high concentration chemistry 40 years later. In subsequent years, other ion P-type pumps are a large, ubiquitous and varied of K+ in the cell, against a higher pumps with similar characteristics were found in many family of membrane proteins that are involved in concentration of Na+ and a different tissues and organisms. They include the sar- many transport processes in virtually all living organ- lower concentration of K+ on coplasmic-reticulum (SR) Ca2+-ATPase that helps to isms. A sequence alignment of four main representa- the outside. control the contraction of skeletal muscle2, the gas- tives of the family (FIG. 1) shows a characteristic pattern tric H+/K+-ATPase that acidifies the stomach, and the of conserved residues, most notably the DKTGTLT H+-ATPase that generates membrane potential in fun- sequence motif (in which D is the reversibly phospho- gal and plant cells3.The biochemical characteristics that rylated Asp). Another characteristic is the presence of are common to these ion pumps are an acid-stable, 10 hydrophobic membrane-spanning helices phosphorylated Asp residue that forms during the (M1–M10; although some have only six or eight), and pumping cycle (the phosphorylated (P) intermediate highly conserved cytoplasmic domains that are gives the family its name) and inhibition by ortho- inserted between helices M2 and M3 and between M4 vanadate, a transition-state analogue. and M5. FIGURE 1 shows that 87 of the roughly 900 Almost 30 years ago4, it was established that the residues are invariant, with identical side chains or Max-Planck Institut für Biophysik, Marie-Curie- P-type ATPases undergo large conformational changes conservative substitutions in equivalent positions. As Straße 13–15, 60439 to translocate ions. Originally, two distinct conforma- the four proteins that are compared in FIG. 1 are from Frankfurt am Main, tions were called E1 and E2 (enzyme-1 and enzyme-2), widely different, unrelated organisms, there is no Germany. with each having a different affinity for the nucleotide doubt that these conserved regions are structurally e-mail: and the transported ions. Later, it became clear that the and functionally significant. Werner.Kuehlbrandt@mpi bp-frankfurt.mpg.de pumping cycle involves several further intermediate This article briefly reviews the distribution of P-type doi:10.1038/nrm1354 states5.Nevertheless, the E1/E2 nomenclature is almost ATPases in biology, then presents a short overview of 282 | APRIL 2004 | VOLUME 5 www.nature.com/reviews/molcellbio © 2004 Nature Publishing Group REVIEWS the different subfamilies and their ion specificities, Box 1 | The Post–Albers cycle before examining in some detail what the recent struc- tures of entire enzymes or individual domains tell us Mg2+–ATP ADP about the fundamental mechanism of ATP-driven ion E1 ion 1 E1–P ion 1 translocation. n n mion 2 mion 2 P-type-ATPase genomics Inside Outside In general, P-type-ATPase genes are more widespread nion 1 nion 1 and varied in eukaryotes than in bacteria and E2 ion 2 E2–P ion 2 archaea. Only four P-type ATPases have been identi- m m fied in Escherchia coli 7.The thermophilic archaean 2+ Methanococcus jannaschii has only one8, and certain par- Pi, Mg asitic bacteria seem to have none7.By contrast, the 9 The general ion-translocation cycle of P-type ATPases Saccharomyces cerevisiae genome has 16 P-type ATPases , (see figure) is based on the Post–Albers scheme for the and no fewer than 45 have been identified in the Na+/K+-ATPase96,97.In simple terms, ion 1 (X+ in FIG. 6) 10 Arabidopsis thaliana genome ,which points to their from the cell interior binds to a high-affinity site in the importance in vascular plants. Genome-wide searches in ATPase E1 state. Ion binding triggers phosphorylation of Caenorhabditis elegans and Drosophila melanogaster have the enzyme by Mg2+–ATP,which leads to the turned up 21 and 15 genes, respectively11.A correspond- phosphorylated E1–P state. The phosphorylated E2–P ing analysis of P-type pumps in the human or mouse state then forms, which is unable to phosphorylate ADP, genome would be interesting, but has not yet been and this state has a reduced affinity for ion 1, which reported. escapes to the outside. Ion 2 (Y+ in FIG. 6) binds from the All P-type ATPases are multi-domain membrane outside and, on hydrolysis of the phosphorylated Asp, proteins with molecular masses of 70–150 kDa. Both the enzyme releases ion 2 to the interior and re-binds ion 1. the carboxyl and amino termini are on the cytoplas- The enzyme is then ready to start another cycle. As a net mic side of the membrane, so they all have an even result of this process, n ions of type 1 are expelled and number of transmembrane segments. Based on m ions of type 2 are imported per molecule of ATP sequence homology, the P-type-ATPase family can be consumed — n and m are small integral numbers divided into five branches (FIG. 2),which are referred between 1 and 3. Pi, inorganic phosphate. to as types I–V.Within these branches, a total of 10 different subtypes or classes can be distinguished7,12. Each subtype is specific for a particular substrate ion the consensus sequence (FIG. 1),but have an extra two (FIG. 2).Functional studies might lead to further rami- helices at the amino-terminal end. Although most fications of the family tree, but it seems unlikely that type-IB ATPases are bacterial, close homologues have entirely new, as yet undiscovered, branches will turn been found in S. cerevisiae 9, plants7 and animals18. up in future genome searches. Mutations in human Cu+-efflux pumps cause the rare,but lethal, hereditary Menkes and Wilson dis- Substrate specificity eases. Because of their medical relevance, these Type-I ATPases. The simplest, and presumably most pumps have received much attention since their dis- ancient, ion pumps are type-I pumps (FIG. 2).Type IA is covery 10 years ago19,20–21. a small class that contains bacterial ion pumps, with the E. coli Kdp K+-pump as the prototype. The Kdp pump Type-IV and -V ATPases. The closest relatives of the is a complex of four different membrane proteins — type-I enzymes are types IV and V (FIG. 2).Type-IV that is, KdpF, KdpA, KdpB and KdpC.The 72-kDa ATPases have so far been found only in eukaryotes, in KdpB subunit contains the catalytic core and ion- which they are involved in lipid transport and the translocation site13 and, with only six membrane-span- maintenance of lipid-bilayer asymmetry. These ‘lipid ning helices, the KdpB subunit is the smallest P-type flippases’ are thought to translocate phospholipids ATPase. from the outer to the inner leaflet of, for example, red- Although most P-type ATPases translocate the small, blood-cell22 and S. cerevisiae 23 plasma membranes. The ‘hard’ cations H+,Na+,K+,Ca2+ and Mg2+, the substrates erythrocyte lipid flippase is a Mg2+-ATPase24.Sequence of type-IB ATPases are ‘soft’-transition-metal ions. comparisons show that they have the main features of Type-IB ATPases — for example, the bacterial metal- the ion-translocating P-type ATPases7,including the resistance proteins CopA14, ZntA15 and CadA16 — ion-binding site in the membrane. In view of the close remove toxic ions such as Cu+,Ag+,Zn2+,Cd2+ or structural homology of the known P-type ATPases, it is Pb2+ from the cell. The homeostasis of indispensable difficult to imagine how the binding site at the centre trace elements, such as Cu+ and Zn2+, is achieved by of a 10-helix bundle can adapt to translocate both ions balancing the activity of these efflux pumps against and phospholipids. Perhaps the lipid flippases are ion ABC-type metal-uptake proteins16,17.Unlike the pumps that work in close association with ion-depen- type-IA ATPases, type-IB ATPases work as single- dent, lipid-transport proteins such as the lipid-translo- chain proteins and have eight membrane-spanning cating ABC transporters22.Even more elusive than the helices.